Use of polypeptides and proteins for the systemic treatment of specific diseases is now well accepted in medical practice. The role that these substances play in therapy is so important that many research activities are being directed towards the synthesis of large quantities by recombinant DNA technology. Many of these polypeptides are endogenous molecules which are very potent and specific in eliciting their biological actions.
A major factor limiting the usefulness of these proteinaceous substances for their intended application is that, when given parenterally, they are eliminated from the body within a short time. This can occur as a result of metabolism by proteases or by clearance using normal pathways for protein elimination such as by filtration in the kidneys. The problems associated with these routes of administration of proteins are well known in the pharmaceutical industry, and various strategies are being used in attempts to solve them.
A peptide family, which has been the focus of much clinical work, and efforts to improve its administration and bio-assimilation, is the interferons. Interferons have been tested in a variety of clinical disease states. The use of human interferon beta, one member of that family, is best established in the treatment of multiple sclerosis. Two forms of recombinant interferon beta, have recently been licensed in Europe and the U.S. for treatment of this disease. One form is interferon-beta-1a (trademarked, sold as AVONEX(copyright), mfg. Biogen, Inc., Cambridge, Mass.) and hereinafter, xe2x80x9cinterferon-beta-1axe2x80x9d or xe2x80x9cIFN-beta-1axe2x80x9d or xe2x80x9cIFN-xcex2-1axe2x80x9d or xe2x80x9cinterferon-xcex2-1axe2x80x9d, used interchangeably. The other form is interferon-beta-1b (trademarked and sold as BETASERON(copyright), Berlex, Richmond Calif.), hereinafter, xe2x80x9cinterferon-beta-1bxe2x80x9d. Interferon beta-1a is produced in mammalian cells using the natural human gene sequence and is glycosylated, whereas interferon beta-1b is produced in E. coli bacteria using a modified human gene sequence that contains a genetically engineered cysteine-to-serine substitution at amino acid position 17 and is non-glycosylated.
Previously, several of us have directly compared the relative in vitro potencies of interferon-beta-1a and interferon beta 1b in functional assays and showed that the specific activity of interferon-beta-1a is approximately 10-fold greater than the specific activity of interferon-beta-1b (Runkel et al., 1998, Pharm. Res. 15: 641-649). From studies designed to identify the structural basis for these activity differences, we identified glycosylation as the only one of the known structural differences between the products that affected the specific activity. The effect of the carbohydrate was largely manifested through its stabilizing role on structure. The stabilizing effect of the carbohydrate was evident in thermal denaturation experiments and SEC analysis. Lack of glycosylation was also correlated with an increase in aggregation and an increased sensitivity to thermal denaturation. Enzymatic removal of the carbohydrate from interferon-beta-1a with PNGase F caused extensive precipitation of the deglycosylated product.
These studies indicate that, despite the conservation in sequence between interferon-beta-1a and interferon-beta-1b, they are distinct biochemical entities and therefore much of what is known about interferon-beta-1b cannot be applied to interferon-beta-1a, and vice versa.
We have exploited the advantages of glycosylated interferon-beta relative to non-glycosylated forms. In particular, we have developed an interferon-beta-1a composition with increased activity relative to interferon-beta-1b and that also has the salutory properties of fusion proteins in general with no effective loss in activity as compared to interferon-beta-1a forms that are not fusion proteins. Thus, if modifications are made in such a way that the products (interferon-beta 1a fusion proteins) retain all or most of their biological activities, the following properties may result: altered pharmacokinetics and pharmacodynamics leading to increased half-life and alterations in tissue distribution (e.g, ability to stay in the vasculature for longer periods of time) Such a formulation is a substantial advance in the pharmaceutical and medical arts and would make a significant contribution to the management of various diseases in which interferon has some utility, such as multiple sclerosis, fibrosis, and other inflammatory or autoimmune diseases, cancers, hepatitis and other viral diseases and diseases characterized by neovascularization. In particular, the ability to remain for longer periods of time in the vasculature allows the interferon-beta-1a to be used to inhibit angiogenesis and potentially to cross the blood-brain barrier.
In particular, the invention relates to an isolated polypeptide having the amino acid sequence X-Y-Z, wherein X is a polypeptide having the amino acid sequence, or portion thereof, consisting of the amino acid sequence of interferon beta; Y is an optional linker moiety; and Z is a polypeptide comprising at least a portion of a polypeptide other than interferon beta. Optional moiety Y and required moiety Z may be linked to either the N- or C-terminus of inteferon beta (X). Preferably, X is human interferon-beta-1a. In the preferred embodiments, Z is at least a portion of a constant region of an immunoglobulin and can be derived from an immunoglobulin of the class selected from IgM, IgG, IgD, IgA, and IgE. If the class is IgG, then it is selected from one of IgG1, IgG2, IgG3 and IgG4. The constant region of human IgM and IgE contain 4 constant regions (CH1, (hinge), CH2, CH3 and CH4, whereas the constant region of human IgG, IgA and IgD contain 3 constant regions (CH1, (hinge), CH2 and CH3. In the most preferred fusion proteins of the invention, the constant region contains at least the hinge, CH2 and CH3 domains. In other embodiments, moiety Z is at least a portion of a polypeptide that contains immunoglobulin-like domains. Examples of such other polypeptides include CD1, CD2, CD4, and members of class I and class II major histocompatability antigens.
Another embodiment of the invention is a fusion protein having an amino terminal region consisting of the amino acid sequence of interferon beta or a portion thereof and having a carboxy terminal region comprising at least a portion of a protein other than interferon beta. The carboxy portion is preferably at least a portion of a constant region of an immunoglobulin derived from an immunoglobulin of the class selected from IgM, IgG, IgD, IgA, and IgE. In the most preferred fusion proteins, the constant region contains at least the hinge, CH2 and CH3 domains.
Another embodiment of the invention is a fusion protein whose interferon beta moiety (e.g., X in the formula above) has been mutated to provide for muteins with selectively enhanced antiviral and/or antiproliferative activity or other advantageous properties relative to non-mutated forms of interferon-beta-1a.
Yet another embodiment of the invention is an isolated DNA encoding for the fusion proteins described above. The invention also pertains to a recombinant DNA comprising an isolated DNA encoding the fusion proteins described above and an expression control sequence, wherein the expression control sequence is operatively linked to the DNA. The scope of the invention also includes host cells transformed with the recombinant DNA sequences of the invention.
The invention further pertains to a method of producing a recombinant polypeptide comprising: providing a population of host cells according to the invention; growing the population of cells under conditions whereby the polypeptide encoded by the recombinant DNA is expressed; and isolating the expressed polypeptide.
A further aspect of the invention is a interferon-beta 1a fusion protein comprising interferon-beta-1a and additional polypeptide with which it is not natively associated, in substantially purified form, the fusion having an antiviral activity that is about equal to the anti-viral activity of interferon-beta-1a lacking the additional polypeptide.
Yet another aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of an interferon-beta-1a fusion protein.
Yet another aspect of the invention is a method of inhibiting angiogenesis and neovascularization using the polypeptides of the invention.